A Distinguishable Role of eDNA in the Viscoelastic Relaxation ofBiofilms

Brandon W. Peterson, Henny C. van der Mei, Jelmer Sjollema, Henk J. Busscher, Prashant K. Sharma

University of Groningen, University Medical Center Groningen, W. J. Kolff Institute, Department of Biomedical Engineering, Groningen, The Netherlands

ABSTRACT Bacteria in the biofilm mode of growth are protected against chemical and mechanical stresses. Biofilms are com-posed, for the most part, of extracellular polymeric substances (EPSs). The extracellular matrix is composed of different chemi-cal constituents, such as proteins, polysaccharides, and extracellular DNA (eDNA). Here we aimed to identify the roles of differ-ent matrix constituents in the viscoelastic response of biofilms. Staphylococcus aureus, Staphylococcus epidermidis,Streptococcus mutans, and Pseudomonas aeruginosa biofilms were grown under different conditions yielding distinct matrixchemistries. Next, biofilms were subjected to mechanical deformation and stress relaxation was monitored over time. A Maxwellmodel possessing an average of four elements for an individual biofilm was used to fit the data. Maxwell elements were definedby a relaxation time constant and their relative importance. Relaxation time constants varied widely over the 104 biofilms in-cluded and were divided into seven ranges (<1, 1 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, and >500 s). Principal-componentanalysis was carried out to eliminate related time constant ranges, yielding three principal components that could be related tothe known matrix chemistries. The fastest relaxation component (<3 s) was due to the presence of water and soluble polysaccha-rides, combined with the absence of bacteria, i.e., the heaviest masses in a biofilm. An intermediate component (3 to 70 s) wasrelated to other EPSs, while a distinguishable role was assigned to intact eDNA, which possesses a unique principal componentwith a time constant range (10 to 25 s) between those of EPS constituents. This implies that eDNA modulates its interaction withother matrix constituents to control its contribution to viscoelastic relaxation under mechanical stress.

IMPORTANCE The protection offered by biofilms to organisms that inhabit it against chemical and mechanical stresses is due inpart to its matrix of extracellular polymeric substances (EPSs) in which biofilm organisms embed themselves. Mechanicalstresses lead to deformation and possible detachment of biofilm organisms, and hence, rearrangement processes occur in a bio-film to relieve it from these stresses. Maxwell analysis of stress relaxation allows the determination of characteristic relaxationtime constants, but the biofilm components and matrix constituents associated with different stress relaxation processes havenever been identified. Here we grew biofilms with different matrix constituents and used principal-component analysis to revealthat the presence of water and soluble polysaccharides, together with the absence of bacteria, is associated with the fastest relax-ation, while other EPSs control a second, slower relaxation. Extracellular DNA, as a matrix constituent, had a distinguishablerole with its own unique principal component in stress relaxation with a time constant range between those of other EPSs.

Received 4 July 2013 Accepted 30 August 2013 Published 15 October 2013

Citation Peterson BW, van der Mei HC, Sjollema J, Busscher HJ, Sharma PK. 2013. A distinguishable role of eDNA in the viscoelastic relaxation of biofilms. mBio 4(5):e00497-13.doi:10.1128/mBio.00497-13.

Invited Editor Matthew Chapman, University of Michigan Editor Scott Hultgren, Washington University School of Medicine

Copyright © 2013 Peterson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unportedlicense, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Address correspondence to Prashant K. Sharma, p.k.sharma@umcg.nl.

Bacteria adhere to virtually all natural and artificial surfaces.Once they adhere to a surface, bacteria rapidly grow into a

biofilm in which they are protected against chemical and mechan-ical stresses. The protection offered by the biofilm to organismsthat inhabit it against chemical stresses, like antibiotic challenges,has been extensively studied (1–3) and is a huge problem in mod-ern medicine, where biofilms account for 65% of all nosocomialinfections in humans, costing over one billion dollars annually totreat in the United States alone (2, 4, 5). Few studies, however,have focused on how bacteria in a biofilm mode of growth copewith mechanical stresses. Oral biofilms on teeth are exposed tocompressive stresses many times per day, especially when growingin fissures (6, 7). Also, intestinal biofilms are exposed to compres-sive stresses during peristaltic bowel movements. Compression of

a biofilm leads to a more compact structure, which is undesirablefrom the perspective of nutrient penetration to deeper layers of abiofilm (8). In addition to compressive stresses, biofilms are sub-jected to tensile stresses. Tensile stresses develop in oral biofilmsduring tooth brushing and may eventually lead to detachment ofbiofilm organisms (9) and their subsequent death in the gastroin-testinal tract. Also, tensile stresses on intestinal biofilms due tofrictional forces arising from stool passage can cause detachmentof biofilm organisms and their removal from their natural envi-ronment. Similar examples hold for other biofilms in the humanbody, as well as for biofilms in many natural and industrial envi-ronments (10).

The EPS (extracellular polymeric substance) matrix in whichbiofilm organisms embed themselves plays an important role in

RESEARCH ARTICLE

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providing protection against chemical and mechanical stresses,which is required for their survival (11). This EPS consists of,among other components, different proteins, polysaccharides,and extracellular DNA (eDNA). Each constituent of the matrixhas specific functions in maintaining overall biofilm health, in-cluding bacterial adhesion and cohesion, retention of water, for-mation of a protective barrier against chemical challenges, sorp-tion of ions and compounds, and exportation of cell components(12). Different bacterial species have their own specific needs andthus require various proportions of matrix constituents in orderto optimize their normal functioning. Pseudomonas aeruginosabiofilms contain copious amounts of eDNA (13) and extracellularpolysaccharides, including alginates in cystic fibrosis pulmonaryisolates (14, 15). Both Staphylococcus aureus and Staphylococcusepidermidis have extracellular proteins in their matrixes (16, 17).Interestingly, only S. aureus could be prevented from formingbiofilms in the presence of DNase I (18). Streptococcus mutans usesextracellular glucans in the presence of sucrose to build its protec-tive matrix (19, 20).

The chemical diversity and adaptability of the EPS matrixamong different bacterial strains are important means throughwhich a biofilm can protect itself against chemical challenges andmechanical stresses. The protection offered by the EPS matrixagainst chemical challenges has been well documented to resultfrom reduced antimicrobial penetration of and adsorption to ma-trix constituents (2, 21–23). Biofilms are both viscous and elasticin nature, enabling bacteria in the biofilm mode of growth tosurvive mechanical stresses (24, 25), but it is unknown how thedifferent biofilm components and matrix constituents contributeto the viscoelastic response of biofilms to mechanical stresses (26).

One way to analyze the viscoelastic response of biofilms to me-chanical stress is stress relaxation measurement. Stress relaxationmeasurement indicates how a biofilm relieves itself from externalstresses, and by using Maxwell analyses (25), the different relax-ation processes that occur in a biofilm under mechanical stress canbe mathematically modeled. Maxwell analyses yield a spring con-stant and a characteristic time constant for each of the relaxationprocesses that occur, but interpretation has seldom gone beyondtheir mathematical background. Tentative interpretations haveattributed the fastest relaxation element to the flow of water inmechanically stressed biofilms, as water has the lowest viscosity ofall biofilm components. On the other hand, the organisms them-selves represent the heaviest masses in a biofilm and their rear-rangement can thus be expected to coincide with the slowest stressrelaxation element. This leaves a wide array of stress relaxationelements with intermediate characteristic time constants that havebeen attributed to the flow of EPS. However, intuition has beenthe only underlying argument for these associations, while theroles of the different constituents of the EPS matrix in stress relax-ation have remained obscure.

Here we have measured compressive stress relaxation of bio-films of different genera (see Table 1) and used a generalized Max-well model with the aim to relate different biofilm componentsand matrix constituents to the different stress relaxation elementsobtained, with a focus on the constituents of the EPS matrix. Bio-films were grown in which specific EPS constituents like polysac-charides, glucans, or eDNA were present, naturally absent, orchemically altered (see Table 1). The total range of characteristicrelaxation time constants observed over 104 different biofilms wasdivided into seven time constant ranges and subjected to a

TABLE 1 Matrix chemistries as chemically determined for biofilms of different species and resulting from different biofilm treatments, includingthe bacterial strains and growth mediums involved in this studya

Treatment, matrix chemistryb Bacterial strainGrowthmediumc

Avg soluble polysaccharideconcn (�g/ml) � SD

Avg eDNA concn(�g/ml) � SD

No treatment, naturally occurring EPS (12) P. aeruginosa SG81 Nutrient broth 134 � 25 130.7 � 5.7No treatment, no naturally occurring EPS

(39)P. aeruginosa SG81-R1 Nutrient broth 28 � 0 49.2 � 7.8

MgCl2, naturally occurring EPS (13, 14) P. aeruginosa SG81 Nutrient broth 76 � 18 70.0 � 3.4DNase I with MgCl2, EPS with less eDNA (18) P. aeruginosa SG81 Nutrient broth 60 � 17 No intact DNA

detectedd

Phosphate-buffered saline, naturally occurringEPS (13, 14)

P. aeruginosa SG81 Nutrient broth 103 � 10 74.6 � 1.8

N-Acetyl-L-cysteine, EPS with lesspolysaccharides (40)

P. aeruginosa SG81 Nutrient broth 68 � 12 68.5 � 8.6

3.0% sucrose added to agar, glucan-rich EPSmatrix (19, 20)

S. mutans ATCC25175

Trypticase soybroth

33 � 9 0.183 � 0.216

No treatment, naturally occurring EPS (41) S. mutans ATCC25175

Trypticase soybroth

4 � 1 0.749 � 0.105

No treatment, naturally occurring EPS (42) S. aureus ATCC 12600 Trypticase soybroth

10 � 1 0.622 � 0.593

No treatment, no EPS matrix (43) S. aureus 5298 Trypticase soybroth

10 � 3 0.150 � 0.068

No treatment, naturally occurring EPS (44) S. epidermidis HBH 45 Trypticase soybroth

12 � 8 5.25 � 2.90

No treatment, no EPS matrix (45) S. epidermidis ATCC12228

Trypticase soybroth

9 � 3 1.62 � 0.93

a Chemical determination was performed in triplicate on biofilms not subjected to deformation.b Chemical treatments were applied to fully grown biofilms, except for the addition of sucrose to the agar growth medium of S. mutans biofilms.c Bacteria were cultured on agar plates with growth medium appropriate for the specific strain (containing 12 g/liter agar).d See Fig. S2 in the supplemental material.

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principal-component analysis that yielded three new principalcomponents that were subsequently related to the chemically de-rived matrix chemistries of the biofilms. A highly distinguishablerole was assigned to intact eDNA as a matrix constituent thatpossesses its own unique principal component with a time con-stant range between those of other EPS constituents. This impliesthat intact eDNA, when present, may interact with other EPS con-stituents to form agglomerates with a unique response to the me-chanical stresses imposed upon a biofilm.

RESULTS

S. aureus, S. epidermidis, S. mutans, and P. aeruginosa (Table 1)were grown on filters, placed on agar plates, and subjected tochemical treatments to yield distinct EPS matrix chemistries, aschemically measured. DNase I treatment was used to break downthe eDNA, and N-acetyl-L-cysteine was used to break down poly-saccharides in the EPS matrix of P. aeruginosa. S. mutans biofilmswere grown with extra sucrose in the growth medium to create aglucan-rich EPS matrix.

Viscoelastic relaxation of compressed biofilms. The biofilmswere compressed to 80% of their original thickness and held in thedeformed state for 100 s. The stress required to keep the biofilmsin the deformed state decreased with time because of differentrearrangement processes in the deformed biofilm (Fig. 1a to c),including flow of EPS matrix constituents and water. The decreasein stress for each biofilm was modeled by using a generalized Max-well model with each element having a spring constant related tothe elastic part of the biofilm and a characteristic relaxation timeconstant related to the ratio of the viscous and elastic parts of thebiofilm. Initially, one Maxwell element was used to fit to the re-laxation curve, after which additional elements were added. FourMaxwell elements generally sufficed to accurately model stressrelaxation of the biofilms, and further addition of elements didnot improve the quality of the fit (Fig. 1d). Biofilms containing anEPS matrix required more Maxwell elements (four or five) to de-

scribe the stress relaxation than biofilms without an EPS matrix(two or three).

Principal-component analysis. The relaxation time constantsof all of the Maxwell elements of the 104 biofilms comprised in thisstudy, taking replicate runs as a separate biofilm, were plotted as afunction of the relative importance of their Maxwell elements(Fig. 2). Relaxation time constants spread over a wide time range,and hence, the total range of relaxation time constants observedover the 104 biofilms investigated was divided into seven relax-ation time constant ranges on a semilog basis as follows: �1, 1 to5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, and �500 s. A principal-component analysis was carried out to determine possible inter-dependence among the different time constant ranges and to re-duce the number of time constant ranges. However, on the basis ofthis division of the total range of relaxation time constants, itoccurred 42 times in the total of 442 Maxwell elements measuredthat one biofilm possessed two data points in one relaxation timeconstant range. Since this impedes principal-component analysis,the semilog-based initial division was slightly adjusted to elimi-nate this redundancy. This led to a new division of relaxation timeconstant ranges (Ci) according to the relaxation time ranges�0.75, 0.75 to 3, 3 to 10, 10 to 25, 25 to 70, 70 to 460, and �460 s,which were subjected to a principal-component analysis. Theprincipal-component analysis yielded three new principal com-ponents (PC1, PC2, and PC3) in terms of coefficients of the seveninitial time ranges to describe the stress relaxation of the differentbiofilms (Fig. 3a), accounting, respectively, for 31, 22, and 15% ofthe variance observed. Incidentally, it was noted that no redun-dancy occurred when the total time constant range was dividedinto a higher number of subranges while yielding similar resultsfor the resulting principal components.

Identification of matrix chemistries and biofilm componentsresponsible for stress relaxation in biofilms. Prominent proper-ties of the biofilms significantly represented in each of the princi-pal components were identified by statistical comparison (Mann-

FIG 1 Panels a to c represent the measured stress relaxation of a P. aeruginosa SG81 biofilm as a function of time, together with model fits to the data, obtainedby using two (panels a and b) or five (panel c) Maxwell elements. Note that panel a extends over 100 s, while panels b and c refer only to the first 5 s of the relaxationprocess. Panel d represents the quality of the fit, indicated by chi-square values, as a function of the number of Maxwell elements used for the fit.

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Whitney U test, P � 0.05; see Fig. S1 in the supplementalmaterial), as related to the strain-specific biofilm properties listedin Table 1. The principal component comprising the two fastestinitial elements (PC1) is negatively impacted by the slowest initialelement (Fig. 3a). Rearrangement of bacteria within a deformedbiofilm can be considered the slowest process, as bacteria consti-tute the heaviest masses. At the same time, water, with dissolvedcomponents, has the lowest viscosity in a biofilm and its flow willform the basis of fast relaxation. Therewith, the process assign-ment to this principal component becomes quite logical, since thepresence of water implies the absence of bacterial cells. The second

principal component (PC2) encompasses intermediate time con-stant ranges (Fig. 3a) of 3 to 10 and 25 to 70 s that are associated bystatistical comparison with EPS (see also Fig. S1). This too is quitelogical, as EPS is a more viscous material than water. Interestingly,the third principal component (PC3) contains only one initialtime constant range that was uniquely associated with the absenceor presence of intact eDNA as an extracellular matrix constituent(Fig. 3a).

DISCUSSION

The viscoelasticity of biofilms reflects their structure and compo-sition and serves, among other functions, to protect a biofilmagainst mechanical and chemical challenges. Little is known, how-ever, about the biofilm components and matrix constituents thatare responsible for the stress relaxation processes within a biofilmas a response to mechanical deformation. In this study, we ana-lyzed the stress relaxation of �100 different biofilms and chemi-cally determined their matrix chemistries. Stress relaxationobeyed a generalized Maxwell model generally comprising four orfive Maxwell elements. Using principal-component analysis, weare the first to establish that three components suffice to describethe viscoelastic relaxation of mechanically deformed biofilms.

The first principal component comprises the fastest two initialtime ranges (�0.75 and 0.75 to 3 s). The fastest time range wasassociated with the flow of water on the basis of its low viscosityand incompressibility. Similar stress relaxation times have beenfound for the cytoplasm of a macrophage (27) under creep andflow of aqueous solutions through micrometer-size channels (25).With water having been associated with the fastest relaxation timeconstant range, the next fastest initial time range could be associ-ated with soluble polysaccharides (Fig. 3b). The second principalcomponent comprises two initial intermediate time ranges (3 to10 and 25 to 70 s) that could be associated with other EPS poly-mers, including glucans (Fig. 3b), with a noticeable separation.

The third principal component is the most interesting one, as itcomprises a single, relatively narrow relaxation time range (10 to25 s) that could be uniquely associated with the presence of intacteDNA as a matrix constituent (Fig. 3b). eDNA as a matrix constit-uent originates from chromosomal DNA and is thought to beproduced through active processes such as autolysis or vesicularsecretion (12, 28, 29). Several recent reports have shown thateDNA is involved in different stages of biofilm formation, includ-ing initial bacterial adhesion, aggregation (12, 30), biofilm archi-tecture (31), and mechanical stabilization of biofilms (29). eDNAperforms its role as a pivotal matrix constituent through acid-baseinteractions with bacterial cell surfaces and polysaccharides (30,32). The ability of eDNA to interact with polysaccharides coin-cides with the position of the third principal component, whichwas found to be uniquely due to intact eDNA, between two initialtime ranges associated with the presence of other EPS polymers.The filamentous structure of eDNA (12) allows it to form agglom-erates with smaller polysaccharides and globular proteins found inthe EPS matrix by means of acid-base interactions. The currenthighly distinguishable role of intact eDNA in the stress relaxationof deformed biofilms suggests that these agglomerates are well-defined structures, otherwise they could not form a single princi-pal component with a narrow time constant range. The narrowrange of the relaxation time constant associated with the presenceof intact eDNA in a biofilm is likely controlled by the length of theDNA strands. Shorter fragments of eDNA strands are more

FIG 2 Relative importance of the individual Maxwell elements of differentbiofilms as a function of their characteristic relaxation time constants in rela-tion to the different matrix chemistries according to Table 1. Each data pointrepresents one Maxwell element with its time constant plotted against its rel-ative importance. Each individual biofilm possessed an average of four or fiveMaxwell elements. Similar biofilms were grown and investigated minimallythree times with separate initial bacterial cultures. Maxwell elements with 0%relative importance have no accompanying time constant and are not plotted,while characteristic time constants exceeding 7,000 s have been assigned avalue of 7,000 s. Vertical lines indicate divisions of relaxation time constantranges (Ci). Panels: a, P. aeruginosa biofilms; b, S. mutans biofilms; c, S. aureusand S. epidermidis biofilms.

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quickly adsorbed onto bacterial cells, while longer fragments tendto have more functional responsibilities (33) and are thus morereadily involved in the formation of agglomerates controllingstress relaxation.

Conclusions. Mechanical stresses lead to deformation andpossible detachment of biofilm organisms, and hence, rearrange-ment processes occur in a biofilm to relieve it from these stressesand maintain its integrity. Maxwell analysis of the stress relaxationof biofilms allows the determination of characteristic relaxationtime constants, but hitherto, biofilm components and matrix con-stituents associated with stress relaxation have never been identi-fied. Using specific bacterial pairs with distinct EPS chemistries aschemically determined, we have, for the first time, related purelymathematical Maxwell elements describing stress relaxation tobiofilm components and matrix constituents on the basis of thecharacteristic relaxation time constants of the Maxwell elementsin terms of three principal components. The presence of water orthe absence of bacteria, for that matter, was associated with thefastest relaxation process, while the rearrangement of other EPSpolymers controls a second, slower relaxation process. Interest-ingly, intact eDNA as a matrix constituent had a distinguishablerole in stress relaxation, with its own unique principal compo-nent. Although several functions of eDNA as a matrix constituenthave been demonstrated in recent years, such a distinguishable

role in stress relaxation is new and adds to the importance ofeDNA in biofilm structure and function.

MATERIALS AND METHODSBiofilm growth and application of chemical treatments. Bacterial strains(Table 1) were stored at �80°C in 7% dimethyl sulfoxide, grown on sheepblood agar plates, and cultured in 10 ml of growth medium (37°C, 17 h).Bacteria were sonicated (10 W, 10 s, 0°C) to disrupt possible aggregatesand enumerated in a Bürker Türk counting chamber. Sterile demineral-ized water (100 ml) and 1 � 108 bacteria were deposited on a membranefilter (0.4-�m pore size, 4.6-cm diameter, HTTP; Millipore, Tullagreen,Carrigtwohill, Ireland) under negative pressure and washed in deminer-alized water (50 ml) for an additional 30 s. Subsequently, the filter with theappropriate growth medium for each bacterial strain (Table 1) was movedonto agar plates containing 12 g/liter Bacto agar (BD, Le Pont de Claix,France) with the bacterial side up and incubated at 37°C for 48 h. Allgrowth media were purchased from Oxoid (Basingstoke, United King-dom), while chemicals were purchased from Sigma (St. Louis, MO) orMerck (Darmstadt, Germany).

Two P. aeruginosa strains were used, SG81 and SG81-R1; SG81-R1 isan isogenic mutant deficient in matrix production. In order to increase ordecrease the prevalence of different constituents in the EPS matrix ofP. aeruginosa SG81, biofilms were subjected to treatment (2 h, 37°C) withphosphate-buffered saline (PBS; 5 mM K2HPO4, 5 mM KH2PO4, 150 mMNaCl, pH 7.0), PBS supplemented with 10 mM MgCl2 and DNase I(0.25 U/ml; Fermentas Life Sciences, Roosendaal, The Netherlands), PBS

FIG 3 (a) Coefficients (aij) of the initial time constant ranges (Ci) for each principal component (PCj) according to the equation

PCj � �i�1

7

�ij � Ei

(see also the last equation in Materials and Methods). (b) Assignment of matrix chemistries to the three principal components (PCj) as distinguished for thedifferent biofilms involved in this study. Principal components are expressed as a function of relaxation time constants based on positive correlations with matrixchemistries defined in Table 1. The matrix chemistries positively associated with PC1 include water and soluble polysaccharides, while the matrix chemistry forPC2 includes other EPS polymers, like insoluble polysaccharides (i.e. glucans). PC3 includes only intact eDNA.

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supplemented with 10 mM MgCl2, or PBS supplemented with 2 mg/mlN-acetyl-L-cysteine. S. mutans biofilms were grown in the absence or pres-ence of sucrose to vary the amount of glucans in the matrix. S. aureusATCC 12600 and strain 5298 and S. epidermidis HBH45 and ATCC 12228were selected as representatives of the genus Staphylococcus for theirknown ability to produce biofilms with or without an EPS matrix, respec-tively. Table 1 summarizes the chemical characteristics of the EPS ma-trixes of the different biofilms grown, as chemically derived in this study.

Soluble-polysaccharide determination. Forty-eight-hour biofilmswere submerged in 5 ml of PBS and vortexed for 1 min, and the resultingfluid was centrifuged (BHG HEKA no. 29380 centrifuge, setting 4,10 min). One milliliter of the supernatant was mixed with 2 ml of an-throne (1 mg/ml in concentrated H2SO4). The samples were allowed toreact for 10 min, and the absorbance at 630 nm was read (SpectronicGENESYS 20) and compared against glucose standards. Final sugar con-centrations were reported in equivalent glucose units (34, 35). Polysac-charide determination was performed in triplicate (Table 1).

eDNA determination. Forty-eight-hour biofilms were submerged in2 ml of eDNA extraction buffer (10 mM EDTA, 0.9% NaCl) and vortexedfor 1 min. Additional buffer was added to P. aeruginosa biofilms to helppellet the matrix material during centrifugation (BHG HEKA no. 29380centrifuge, setting 4, 10 min). Dilutions were made for the supernatant inextraction buffer, and 1 ml of solution was combined with 500 �l ofphenol (8.3 g/ml) and 500 �l of chloroform. After centrifugation (2,700 �g, 5 min, 10°C), the aqueous layer was collected and an additional 500 �lof chloroform was added before centrifugation (2,700 � g, 5 min, 10°C).Two 700-�l aliquots of the aqueous layer were mixed with 140 �l of 3 Msodium acetate and 460 �l of isopropyl alcohol. The aliquots were centri-fuged (15,300 � g, 20 min, 10°C), 690 �l of the aqueous layer was re-moved, and 500 �l of 100% ethanol was added. The samples were centri-fuged (15,300 � g, 15 min, 10°C), and the liquid phase was removed,leaving 50 to 100 �l to evaporate overnight. Samples were reconstitutedwith 45 �l of extraction buffer (4 h, 20°C). The two aliquots were recom-bined, forming one sample, and treated with 4 �l of RNase A (20 mg/ml,30 min, 37°C) (36). Samples were analyzed with the CYQuant kit (Molec-ular Probes) for fluorescence intensity against DNA standards (480 and520 nm; FLUOstar OPTIMA plate reader). eDNA determination was per-formed in triplicate (Table 1).

In order to verify whether the analysis was pertinent to intact eDNA,biofilms of P. aeruginosa SG81 prior to and after treatment with MgCl2and DNase I were run on a 1% agarose gel for 90 min at 65 W (see Fig. S2in the supplemental material).

Biofilm compression and analysis of viscoelastic relaxation. Bio-films were deformed with a low-load compression tester (37). Briefly, astainless steel plunger (diameter, 0.25 cm) was lowered toward a samplestage and the position of the stage was recorded. Next the plunger waslowered toward the top of the biofilm until a touch load of 0.01 g wasachieved and its position was recorded again. The difference betweenplunger positions determined the thickness of the biofilms. Next, the bio-films were deformed 20% (strain 0.2) in 1 s and the deformation wassubsequently held constant for 100 s while stress development was mon-itored over time (38). Stress relaxation as a function of time, E(t), wasfitted by using a generalized Maxwell model according to the equation

E(t) � E1e�t ⁄ �1 � E2e

�t ⁄ �2 � E3e�t ⁄ �3 � � Eie

�t ⁄ �i

where E(t) is the total stress divided by the induced strain expressed asthe sum of i Maxwell elements with a spring constant Ei and characteristicrelaxation time �i. Model fitting was performed with the Microsoft Excel2007 Solver module without imposing any restrictions on the value of Ei

or �i, except that the value had to be positive to maintain its physicalrelevance and �i had to be �0.01 s. Initially, one Maxwell element wasused to fit to the stress relaxation data and then additional elements wereadded until no further decrease in chi-squared values was observed(Fig. 1). For each biofilm, a relative importance was assigned to eachelement on the basis of the value of its spring constant, Ei, and expressed as

its spring constant’s percentage of the sum of all of the elements’ springconstants at t � 0 according to the equation

Ei �Ei

�i�1

7Ei

Identification of biofilm components and matrix constituents thatinfluence the viscoelastic deformation of compressed biofilms. The firststep in the identification of biofilm components and matrix constituentsthat influence the viscoelastic relaxation of compressed biofilms was todivide the total range of relaxation time constants observed over the 104biofilms investigated into seven initial relaxation time constant ranges ona semilog basis as follows: �1, 1 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500,and �500 s. On the basis of the relative importance, E� i, of the data in eachtime range, a principal-component analysis (SPSS v. 16.0 for Windows,data reduction factor analysis, principal-component analysis with a max-imum of 25 iterations) was carried out to identify which combinations ofrelaxation time constant ranges could explain the variance in the data setbest. The resulting new principal components (PCj) comprised coeffi-cients from the seven initial relaxation time ranges according to the equa-tion

PCj � �i�1

7

aij � Ei

for j � 1 to 3 and in which E� i is the relative importance of the springconstants in each initial time constant range i and aij, the correspondingcoefficients. The value for each principal component was calculated byusing the equation above.

Next, median values of the principal components for each stress relax-ation experiment were calculated according to the equation above. Resultsfor bacterial pairs with known differences in their matrix chemistry werecompared by using a Mann-Whitney U test. Whenever median values forbiofilms with a specific known matrix chemistry were significantly higherthan data for biofilm lacking that specific chemistry at a level of P � 0.05,the chemistry was related to a specific principal component, PCj (see alsoFig. S1 in the supplemental material).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at http://mbio.asm.org/lookup/suppl/doi:10.1128/mBio.00497-13/-/DCSupplemental.

Figure S1, TIF file, 0.8 MB.Figure S2, TIF file, 5.8 MB.

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